Noninvasive imaging and characterization of engineered tissues would greatly enable the development, testing, and commercialization of new therapies. Ultrasound imaging and microscopy have been used to characterize some aspects of tissue structure, but conventional ultrasound techniques are hampered by instrument-dependence and are limited to qualitative assessment of morphology. The objective of this two-year exploratory project is to develop a quantitative imaging technique for the rapid and high resolution characterization of the composition, structure and mechanical properties of mineralized engineered tissues. It applies high frequency spectral ultrasound imaging (SUSI) to create high resolution images and power spectra of mineral-containing tissues. Parametric analysis of the power spectra provides information on the location, density, and particle size of the mineral component. Augmentation of SUSI with acoustic radiation force (ARF) elastography allows measurement of the local and bulk mechanical properties. The project has three Specific Aims (SA). In SA1 we will demonstrate the use of SUSI to quantitatively characterize the spatiotemporal development of a mineral phase in three dimensional (3D) engineered orthopaedic tissues. In SA2 we will implement ARF elastography for noncontact measurement of tissue mechanical properties in 3D engineered orthopaedic tissues. Finally, in SA3 we will correlate the composition and structure information from SA1 with the mechanical property data from SA2. Replacing the currently used destructive biochemical and histological methods of tissue characterization with noninvasive, spatiotemporal characterization of tissue development would greatly enhance research in tissue engineering. In addition, such methods would facilitate quality control and monitoring of engineered tissue products. The ability to correlate composition and mechanical properties in developing tissues over time also could lead to new insights into structure-function relationships and mechanobiology.